Amplification of signal using a bead-based oligonucleotide assay

Abstract

The present invention regards amplification of a signal from a hybridization-based oligonucleotide assay. In some embodiments, a bead comprises an oligonucleotide hybridized to a labeled polynucleotide from a sample, and a signal generated from a complex thereof is amplified through labeled antibodies directed to a receptor for the label. In particular embodiments, the assay provides information on gene expression.

1 . A method for amplifying a signal for detection of a polynucleotide, comprising the steps of: 
 (a) providing at least one microsphere linked to at least one pre-optimized oligonucleotide;    (b) hybridizing a labeled target polynucleotide to said oligonucleotide to form an oligonucleotide/target polynucleotide complex, wherein said complex comprises a detectable signal through the binding of a receptor to the label; and    (c) providing a labeled ligand for said receptor, wherein when said ligand binds said receptor, said signal is amplified.    
     
     
         2 . The method of  claim 1 , wherein the pre-optimized oligonucleotide is selected with an algorithm.  
     
     
         3 . The method of  claim 2 , wherein said algorithm utilizes at least one of the following selection criteria: 
 (a) selecting at least one perfect match pre-optimized oligonucleotide, wherein the selected at least one perfect match pre-optimized oligonucleotide has an acceptable measure of correlation with a standard gene expression value;    (b) selecting at least one perfect match and minus mismatch pre-optimized oligonucleotide pair, wherein within a pair the selected at least one perfect match pre-optimized oligonucleotide minus the mismatch pre-optimized oligonucleotide has an acceptable measure of correlation with a standard gene expression value;    (c) selecting at least one pair of pre-optimized oligonucleotides from different pre-optimized oligonucleotide sets, wherein the ratio of signals in the pre-optimized oligonucleotides in the at least one pair of pre-optimized oligonucleotides has an acceptable correlation with a standard signal ratio; and    (d) selecting at least one perfect match pre-optimized oligonucleotide, wherein the perfect match pre-optimized oligonucleotide has an acceptable relative standard deviation.    
     
     
         4 . The method of  claim 1 , wherein said pre-optimized oligonucleotide is further defined as being selected by the steps of: 
 providing a sample comprising at least one target polynucleotide;    subjecting said sample to an array of oligonucleotides, wherein the hybridization of said target polynucleotide to at least one oligonucleotide in the array provides a detectable hybridization fingerprint; and    identifying at least one optimal oligonucleotide from said fingerprint.    
     
     
         5 . The method of  claim 1 , wherein said pre-optimized oligonucleotide is further defined as being selected by the steps of: 
 providing a sample comprising a plurality of target polynucleotides, said target polynucleotides defined as RNA polynucleotides from more than one gene;    subjecting said sample to an array of oligonucleotides, wherein the hybridization of more than one different RNA polynucleotide to a respective oligonucleotide in the array provides a detectable hybridization fingerprint for more than one gene; and    identifying at least one optimal oligonucleotide for said more than one gene from said fingerprint.    
     
     
         6 . The method of  claim 1 , wherein said identifying step utilizes an algorithm to identify said oligonucleotide.  
     
     
         7 . The method of  claim 6 , wherein said algorithm identifies an oligonucleotide having complete complementarity to at least a portion of said target polynucleotide.  
     
     
         8 . The method of  claim 1 , wherein the target polynucleotide is comprised in a plurality of RNA polynucleotides and the concentration of said plurality is from about 1 μg to about 10 μg.  
     
     
         9 . The method of  claim 1 , wherein said ligand comprises an antibody.  
     
     
         10 . The method of  claim 1 , wherein the label of the target polynucleotide and/or the label of the ligand comprises a fluorescent label, an enzyme label, a chemical label, or a gold label.  
     
     
         11 . The method of  claim 1 , wherein the label of the target polynucleotide and the label of the ligand are identical.  
     
     
         12 . The method of  claim 1 , wherein said microsphere is comprised in a plurality of microspheres and said target polynucleotide is comprised in a plurality of RNA polynucleotides.  
     
     
         13 . The method of  claim 12 , wherein the plurality of RNA polynucleotides is comprised in a mRNA-containing sample, and said method is further defined as a method for providing mRNA expression profiling information.  
     
     
         14 . The method of  claim 12 , wherein at least one microsphere in said plurality of microspheres comprises different oligonucleotides from the oligonucleotides of another microsphere in said plurality.  
     
     
         15 . The method of  claim 12 , wherein at least one microsphere in the plurality comprises more than one non-identical pre-optimized oligonucleotide having sequence complementary to the same RNA polynucleotide.  
     
     
         16 . A composition, comprising: 
 a plurality of microspheres, each microsphere linked to at least one pre-optimized oligonucleotide, wherein said oligonucleotide is hybridized to a labeled RNA polynucleotide forming an oligonucleotide/labeled RNA polynucleotide hybridized complex, and wherein said complex comprises a detectable signal through the binding of a receptor to the label, said signal amplified upon binding of a labeled ligand for the receptor.    
     
     
         17 . The composition of  claim 16 , wherein at least one microsphere in said plurality of microspheres comprises different oligonucleotides from the oligonucleotides of another microsphere in said plurality.  
     
     
         18 . The composition of  claim 16 , wherein at least one microsphere in the plurality comprises more than one non-identical pre-optimized oligonucleotide each having sequence complementary to the same RNA polynucleotide.  
     
     
         19 . A method of optimizing an oligonucleotide hybridization-based assay, comprising the steps of: 
 providing a sample comprising at least one target polynucleotide;    subjecting said sample to an array of oligonucleotides, wherein the hybridization of said target polynucleotide to at least one oligonucleotide in the array provides a detectable hybridization fingerprint;    identifying at least one optimal oligonucleotide from said fingerprint, wherein said identifying step utilizes an algorithm defined by at least one of the following selection criteria: 
 (a) selecting at least one perfect match pre-optimized oligonucleotide, wherein the selected at least one perfect match pre-optimized oligonucleotide has an acceptable measure of correlation with a standard gene expression value;  
 (b) selecting at least one perfect match and minus mismatch pre-optimized oligonucleotide pair, wherein within a pair the selected at least one perfect match pre-optimized oligonucleotide minus the mismatch pre-optimized oligonucleotide has an acceptable measure of correlation with a standard gene expression value;  
 (c) selecting at least one pair of pre-optimized oligonucleotides from different pre-optimized oligonucleotide sets, wherein the ratio of signals in the pre-optimized oligonucleotides in the at least one pair of pre-optimized oligonucleotides has an acceptable correlation with a standard signal ratio; and  
 (d) selecting at least one perfect match pre-optimized oligonucleotide, wherein the perfect match pre-optimized oligonucleotide has an acceptable relative standard deviation; and  
   subjecting said optimal oligonucleotide to an oligonucleotide hybridization-based assay.